Pichia is a methylotrophic yeast that is widely used for the production of heterologous proteins of industrial and academic interest (Cregg, 1998; Higgins and Cregg, 1998). FLD is an important enzyme in the utilization of methanol as a carbon and energy source (Veenhuis et al., 1983). In methylotrophic yeasts, the methanol metabolic pathway is thought to be nearly the same, beginning with the oxidation of methanol to formaldehyde by alcohol oxidase (AOX), a hydrogen peroxide-producing oxidase that is sequestered in an organelle called the peroxisome. Hydrogen peroxide is then degraded to oxygen and water by catalase, the classic peroxisomal marker enzyme. A portion of the resulting formaldehyde condenses with xylulose-5xe2x80x2-monophosphate in a reaction catalyzed by dihydroxyacetone synthase (DAS), the third peroxisomal methanol pathway enzyme. The products of this reaction, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone, then leave the peroxisome and enter a cyclic pathway that regenerates xylulose-5xe2x80x2-monophosphate and also generates one net molecule of GAP for every three turns of the cycle. GAP is used for biosynthesis of carbon skeletons for cell growth. Another portion of the formaldehyde leaves the peroxisome and is oxidized to formate by formaldehyde dehydrogenase (FLD) and then to carbon dioxide by formate dehydrogenase (FDH). Both of these reactions produce reducing power in the form of NADH. One model of FLD function is that the NADH generated by FLD and FDH serves as the primary source of energy during growth on methanol (Veenhuis et al., 1983). The second model proposes that most energy for methanol growth comes from the oxidation of one or more of the xylulose-5xe2x80x2-monophosphate cycle intermediates by tricarboxcylic acid cycle enzymes, and that the primary role of FLD is to protect the cell from toxic formaldehyde that accumulates with excess methanol in the medium (Sibirny et al., 1990).
In addition to methanol, FLD is also involved in the metabolism of certain methylated amines (e.g. methylamine and choline) as sole nitrogen sources (Zwart et al., 1980). In this pathway, amine groups are first liberated by a peroxisomal amine oxidase, leaving formaldehyde which is further oxidized by FLD and FDH. When growing on methylamine as sole nitrogen source, high levels of FLD are induced even in the presence of excess glucose. Thus, the primary role of FLD in methylamine metabolism appears to be for protecting cells from the toxic effects of formaldehyde and not for generating carbon or energy.
FLD synthesis is regulated independently in response to either methanol as sole carbon source and energy source or to methylamine as sole nitrogen source. Thus, for example, only low levels of FLD are observed in cells growing on glucose- and ammonium ion-containing medium, whereas on either methanol-ammonium ion or glucose-methylamine media, FLD levels are high.
In the Pichia system, most foreign genes are expressed under the transcriptional control of the P. pastoris alcohol oxidase 1 gene promoter (PAOX1), the regulatory characteristics of which are well suited for this purpose. The promoter is tightly repressed during growth of the yeast on most common carbon sources, such as glucose, glycerol, or ethanol, but is highly induced during growth on methanol (Tschopp et al., 1987; U.S. Pat. No. 4,855,231 to Stroman, D. W., et al). For production of foreign proteins, PAOX1-controlled expression strains are initially grown on a repressing carbon source to generate biomass and then shifted to methanol as the sole carbon and energy source to induce expression of the foreign gene. One advantage of the PAOX1 regulatory system is that P. pastoris strains transformed with foreign genes whose expression products are toxic to the cells can be maintained by growing under repressing conditions.
Although many proteins have been successfully produced using PAOX1, this promoter is not appropriate or convenient in all settings. For example, in shake-flask cultures, methanol rapidly evaporates, and it is inconvenient to monitor methanol concentrations and repeatedly add the compound to the medium. In addition, the storage of large amounts of methanol needed for the growth and induction of PAOX1-controlled expression strains in large-volume high-density fermentor cultures is a potential fire hazard. There is a need therefore, for an alternative promoter to PAOX1, which is both transcriptionally efficient and regulatable by a less volatile and flammable inducer. The present invention provides the P. pastoris and Hansenula polymorpha formaldehyde dehydrogenase gene (FLD) promoter having both properties.
In addition, there is a need for a selectable marker which functions in methylotrophic yeasts other than a selectable marker which is an antibiotic resistance gene. At present, only the ZeoR gene can be used to transform into P. pastoris strains independent of their genotype. In addition, ZeoR is the only that gene can be used to directly select for P. pastoris strains that receive multiple copies of an expression vector (by increasing the concentration of zeocin in selective medium). A second gene which confers resistance to the antibiotic G418 (G418R) can be used to screen for multicopy expression strains of P. pastoris but its use requires that an auxotrophic/biosynthetic gene selection marker must also be included in vectors to select for transformants. The FLD structural gene of the present invention may be used as a selectable marker in methylotrophic yeast cells and does not confer resistance to antibiotics.
The present invention is directed to isolated nucleic acid sequences comprising a formaldehyde dehydrogenase gene (FLD) from methylotrophic yeasts. In one embodiment of the invention, the isolated nucleic acids comprise sequences which hybridize under low stringency conditions to at least one of the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:5, or a sequence complementary to the sequence set forth in SEQ ID NOs: 1 or 5.
Also provided is an FLD gene from Pichia pastoris (FLD1) having the restriction map set forth in FIG. 7 and an FLD gene from Hansenula polymorpha having the restriction map shown in the cross hatched area of FIG. 10.
In one embodiment of the invention, there is provided an isolated nucleic acid comprising an FLD gene from a methylotrophic yeast with a coding sequence having a sequence homology of about 70% to about 85% when compared to the nucleotide sequence set forth in SEQ ID NO:5. In another embodiment of the invention, there is provided an isolated nucleic acid comprising an FLD gene from a methylotrophic yeast with a coding sequence having a sequence homology of about 85% to about 95% when compared to the nucleotide sequence set forth in SEQ ID NO:5. In still another embodiment, there is provided an isolated nucleic acid comprising an FLD gene from a methylotrophic yeast with a coding sequence having a sequence homology of greater than about 95% when compared to the nucleotide sequence set forth in SEQ ID NO:5. Isolated nucleic acids comprising the sequences set forth in SEQ ID NO:1 or SEQ ID NO:5 are also provided.
The present invention also provides an isolated nucleic acid from a methylotrophic yeast comprising an FLD promoter. The promoter is located upstream from the translational start codon of an FLD gene having a coding sequence with a sequence homology of about 70% to about 85% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5. In another embodiment, there is provided an isolated nucleic acid from a methylotrophic yeast comprising an FLD promoter from an FLD gene having a coding sequence with a sequence homology of about 85% to about 95% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5. In a preferred embodiment, the promoter is from an FLD gene having a coding sequence with a sequence homology of greater than about 95% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5. Particularly exemplified is a Pichia pastoris FLD1 promoter comprising the sequence set forth in SEQ ID NO:3.
Also in accordance with the present invention, there is provided an isolated nucleic acid comprising an FLD 3xe2x80x2 termination sequence from a methylotrophic yeast. The 3xe2x80x2 termination sequence is located downstream from the translational stop codon of an FLD gene having a coding sequence with a sequence homology of at about 70% to about 85% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5. In another embodiment of the invention, there is provided an isolated nucleic acid comprising an FLD 3xe2x80x2 termination sequence from a gene having a coding sequence with a sequence homology of at about 85% to about 95% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5. In a preferred embodiment of the invention, there is provided an isolated nucleic acid comprising an FLD 3xe2x80x2 termination sequence from a gene having a coding sequence with a sequence homology of greater than about 95% when compared to the sequence set forth in SEQ ID NO:5.
Also provided are isolated nucleic acids comprising an FLD gene wherein said FLD gene encodes a product having an amino acid sequence identity of about 30% to about 49%, or about 50% to about 90%, or greater than about 90% when compared to the amino acid sequence as set forth in SEQ ID NO:2.
In addition, the present invention also provides an isolated nucleic acid comprising at least one of a promoter, coding sequence or 3xe2x80x2 termination sequence from an FLD gene wherein said FLD gene encodes a product having an amino acid sequence identity of about 30% to about 49%, or about 50% to about 90%, or greater than about 90% when compared to the amino acid sequence as set forth in SEQ ID NO:2.
In addition, the present invention provides expression cassettes, vectors and host cells comprising the subject isolated nucleic acids.
Also in accordance with the present invention, there is provided a method for directing expression of a heterologous gene in a methylotrophic yeast. The method comprises introducing into a methylotrophic yeast cell an isolated nucleic acid comprising an FLD promoter isolated from a methylotrophic yeast, said promoter operably linked at its 3xe2x80x2 end to the 5xe2x80x2 end of a heterologous gene, said heterologous gene operably linked at its 3xe2x80x2 end to the 5xe2x80x2 end of a termination sequence which functions in a methylotrophic yeast. The methylotrophic yeast cells are grown in a medium having a suitable carbon source such as glycerol or glucose and having a suitable nitrogen source such as an ammonium salt or ammonium hydroxide. After the carbon or nitrogen source is depleted, expression of said heterologous gene is induced by addition of methanol or methylamine or both methanol and methylamine. Expression may also be induced by the addition of formaldehyde, formate, or a methylated amine.
A method for selecting a formaldehyde resistant host cell is also provided by the present invention. The method comprises transforming a methylotrophic yeast cell with a vector comprising an FLD gene, said FLD gene operably linked at its 5xe2x80x2 end to an FLD promoter or a heterologous promoter which functions in said yeast cell, said FLD gene operably linked on its 3xe2x80x2 end to a 3xe2x80x2 termination sequence which functions in said yeast cell. Host cells are grown in the presence of formaldehyde and a yeast cell which grows in the presence of formaldehyde is selected.
The present invention also provides a strain of methylotrophic yeast which is defective in an FLD gene (fld) such as Pichia pastoris GS241 (fld1-1). Also provided is a strain of methylotrophic yeast which is defective in an FLD gene and auxotrophic for another biosynthetic gene.
In accordance with the present invention, a kit is provided which comprises an expression cassette comprising an FLD promoter and a 3xe2x80x2 termination sequence which functions in a methylotrophic yeast. At least one restriction site is located between the FLD promoter and 3xe2x80x2 termination sequence so that a heterologous gene may be inserted and operably linked to the promoter and the 3xe2x80x2 termination sequence. Also included in the kit is a vector which either replicates in a methylotrophic yeast or which integrates into the genome of a methylotrophic yeast, which vector comprises a marker gene and one or more restriction sites for insertion of the expression cassette.
In addition, the present invention provides a kit which comprises an expression vector comprising an FLD gene as a selectable marker gene and an expression cassette. The expression cassette comprises a promoter and a 3xe2x80x2 termination sequence which functions in a methylotrophic yeast, and has at least one restriction site located between the promoter and 3xe2x80x2 termination sequence so that a heterologous gene may be inserted and operably linked to the promoter and said 3xe2x80x2 termination sequence.